Method of determining haemoglobin

ABSTRACT

METHOD FOR THE DETERMINATION OF THE HAEMOGLOBIN COMTENT IN BLOOD. A BLOOD SAMPLE IS MIXED WITH A SOLUTION OF PERCHORIC ACID IN ACETIC ACID AND THE MIXTURE IS CHECKED FOR COMPLETE NEUTRALIZATION OF THE PERCHLORIC ACID ADDED. THE AMOUNT OF PERCHLORIC ACID USED IS SELECTED IN SUCH A WAY THAN AN ARBITRARY PRE-SELECTED AMOUNT OF HAEMOGLOBIN IS NEUTRALIZED. TO DETERMINE COMPLETENESS OF THE NEUTRALIZATIONL, A COLOR INDICATION MAY BE USED ADVANTAGEOUSLY.

y 18, 1972 E. PRIDE EI' L 3,677,707

METHOD OF DETERMINING HAEMOGLOBIN Filed Oct. 27, 1969 2 Sheets-Sheet 1 14 I5 methaemoglobm method |2 I3 Haemoglobin concentratlon In g/IOO ml by Cyan Perchloric Acid Titre: Haemoglobin Content (Cyanmethaemoglobin) poo q M 01 ua wnnba p nv opo qzua 6m I July 18, 1972 PRIDE EI'AL METHOD OF DETERMINING HAEMOGLOBIN 2 Sheets-Sheet 2 Filed Oct. 27, 1969 NQE $83 go 5 28 5:. EEC c5235; 25 22 23 2 poolq 1m; 01 wanmnba puv ouo qola 6w United States Patent US. Cl. 23-230 B Claims ABSTRACT OF THE DISCLOSURE Method for the determination of the haemoglobin content in blood. A blood sample is mixed with a solution of perchoric acid in acetic acid and the mixture is checked for complete neutralization of the perchloric acid added. The amount of perchloric acid used is selected in such a way that an arbitrary pre-selected amount of haemoglobin is neutralized. To determine completeness of the neutralization, a colour indication may be used advantageously.

This invention relates to method and apparatus for the determination of the haemoglobin content of blood. The determination is carried out by a test designed to give a qualitative assessment of the haemoglobin content of a blood sample by a simple colour change in the reactants employed including the test blood sample. Primarily the test is for human blood but with some minor changes can be employed with blood samples with other mammals. The main purpose of the test is to screen blood samples for detection of anaemia by means which give positive or negative results, so that it can be carried out quickly by general medical practitioners in their routine examination and treatment of patients in their consulting oflices. The test may also be directed to detecting individuals who have a haemoglobin content above or below any selected level.

The condition of iron deficiency anaemia in mammals especially in humans is well known. It is generally defined as a subnormal level of haemoglobin in the blood. The accepted 100% value is 14.8 g. haemoglobin per 100 ml. blood, although a variation between 12 and 16 g./ 100 ml. in adult males and 11 to 15 g./l00 ml. in adult females is considered normal. Blood with haemoglobin level below 12 g./100 ml. is generally considered to be anaemic. The iron found in the blood is mainly in the haemoglobin: the normal range of iron levels for whole blood is 40 to 55 mg. Fe/ 100 ml., of which only 0.08 to 0.2 mg. Fe/il00 ml. is present in the serum, the remainder being in the haemoglobin. Since the iron content of the serum is negligible, a measurement of the iron content of whole blood can be used to estimate the haemoglobin concentration, using the molecular formula of haemoglobin (C7 H 1 FeN 203O2o S2)4 in the Patient from a sample under test is taken.

In existing methods of determining the haemoglobin content of blood it is known to convert the haemoglobin to various derivatives, e.g., alkaline haematin, acid haematin, cyanmethaemoglobin, carboxyhaemoglobin or oxyhaemoglobin, followed by light absorption measurements at defined wavelengths and comparing them against a standard. It was not found possible to adapt these methods to a limit test as they all rely on some form of colour matching against standardized blood samples or synthetic standards which is inaccurate without specialized equipment. Iron in blood has been estimated by forming colour complexes after ashing of the blood and by direct titration. The nature of these procedures are such that they are not practicable in a doctors consulting ofiice nor are they adaptable as a limit test. One limit test for haemoglobin 3,677,707 Patented July 18, 1972 is the copper sulphate test which gives an indication of the specific gravity of the blood which can then be related to the haemoglobin concentration. The test is used for preliminary screening by the Blood Transfusion Service in the United Kingdom but highly reproducible and precise technique is required to obtain good results.

During the search for possible tests, it was found that haemoglobin could be titrated as a base in non-aqueous media with perchloric acid.

The main object of the present invention is the provision of a simple test for the detection of the presence of a specific amount of haemoglobin in whole blood; it is another object of this invention to provide a haemoglobin test which is simple to carry out by the physician in his oflice; it is a further object of this invention to provide a fast, reproducible test for the determination of the presence or absence of a specified amount of haemoglobin in a small sample of whole blood; it is a still further object of this invention to provide a test kit for carrying out the simple and fast determination of the presence or absence of a specified amount of haemoglobin in blood.

These and other objects are accomplished by providing a method for determining haemoglobin in blood comprising admixing a sample of whole blood with a solution of perchloric acid in glacial acetic acid, the amount of perchloric acid present corresponding to that amount necessary for the complete neutralization of a selected haemoglobin content of whole blood, and determining whether or not complete neutralization of the haemoglobin in the blood sample under test has occurred by means of an indicator contained in the solution. The size of the blood sample and the amount of the perchloric acid used in each individual test can be selected in such a manner that the indicator shows the presence or absence of any preselected level of haemoglobin in the blood sample and the test can easily be expanded to produce a quantitative limit reading of the haemoglobin content. For example, the test solution can be prepared to contain that concentration of perchloric acid which when mixed with 0.02 part of whole blood per 10 parts of test solution shows an indicator change at 12.35 :0.35 g./ ml. of blood. With the same solution, the change of the indicator can be set to indicate the presence or absence of 1482:0335 g./100 ml. of the same blood sample by using a greater volume of the test reagent solution, the volume being calculated by means of the equation given in Example 2. The same can also be accomplished by reducing the size of the blood sample to a volume which can be calculated from the equation given in Example 2. In this fashion the invention may by used to detect individuals who have a haemoglobin content above or below any particular value.

The blood volume may vary from 0.5 microlitres to 10 ml.; the ratio of blood volume to volume of glacial acetic acid may vary from 1:2,500 to 1:125; the concentration of perchloric acid in glacial acetic acid may vary from 0.001% w./v. to 0.02% w./v. It will be understood that by suitable selection from the aforesaid volume ratios and concentrations it is possible to indicate haemoglobin content at any required level. Moreover, the amount of glacial acetic acid employed is not critical.

Suitable indicators for the test are those classified as colour indicators for non-aqueous titrations. In particular C.I. Solvent Blue 19' and a component thereof (Abbott Blueto be herein defined) were found particularly suitable.

In a general embodiment of the present invention, a known volume of standardized perchloric acid in glacial acetic acid containing Abbott Blue indicator may be used. The amount of perchloric acid will be neutralized, as evidenced by the mid-point colour of the indicator, by a level of haemoglobin corresponding to the amount of haemoglobin for which the test solution, its volume and the volume of blood are pre-set. In a specific embodiment, 10 ml. of a test solution containing 640 l. of perchloric acid and Abbott Blue in glacial acetic acid shows the colour change with 0.02 ml. of blood occurring over a range of 12.0-12.7 g. haemoglobin/100 ml. Therefore, normal blood samples having a haemoglobin level in excess of 12.7 g./ 100 ml. will react with all the perchloric acid and an excess of haemoglobin will be present resulting in a blue colouration. If the haemoglobin level is below 12.0 g./100 ml., excess perchloric acid will remain and the solution will remain pink. Sensitivity trials with this colour change (in the system perchloric acid 0.32- 0.64 mg./glacial acetic acid approximately 10 ml./Abbott Blue 0.1 mg.) showed that a diiference of 0.7 g. haemoglobin per 100 ml. blood could be detected fairly easily.

In order that the invention may be more fully understood some embodiments in accordance therewith will now be described, reference being made to the accompanying drawings and the following examples:

FIG. 1 is a graph showing the results of perchloric acid titres on a number of blood samples showing the perchloric acid content as ordinate and the haemoglobin concentration as abscissa, using the cyanmethaeoglobin method; and

FIG. 72 is a similar graph to FIG. 1 with the haemoglobin concentration deduced from the iron content of the blood samples.

EXAMPLE 1 Serum, from a number of blood samples of known and widely diifering haemoglobin levels, was titrated with perchloric acid in glacial acetic acid. The serum titre per unit volume of blood Was found to be constant.

Precise correlation of the perchloric acid titre with haemoglobin level was established from the results depicted in Table 1 and their graphical expression in FIG. 1 and FIG. 2. It was found that a sample of Whole blood containing 0.1235 g. haemoglobin is equivalent to 32 mg. perchloric acid.

TABLE 1 (B) (C) Haemoglobin Haemoglobin calculated by eyanmet- (D) .Mg; from iron haemoglobin perchloric (AL-Fe; level, method, acid=i ml. Number mgJlOO ml. g./100 ml. g./100 ml. blood 1 49.06 14. 27 14.6 35. 8 2 47.84 13. 90 14. 3 34. 9 3.. 42. 75 12.44 12. 5 33. 4 55. 37 16. 11 16. 2 37. 0 5- 46. 20 13.44 12. 8 33.6 6 37.45 10. 89 16. 2 37. 4 7 48. 65 14. 16 14. 5 34. 3 8.. 55. 17 16. 04 16. 2 37. 0 9- 48.65 14.16 14. 7 34. 2 10 42. 95 12. 49 12. 5 32. 8 11 51. 70 15. 04 15. 4 35. 2 12 1 37. 26 1 10.83 14. 7 35. 0 13 27. 28 7. 93 8. 8 27. 8 14 51.50 14.98 14. 9 35.4 15 46. 13. 44 13. 9 34. 0 16 48. 65 14. 16 14. 7 34. 2 17 44. 99 13. 08 13. 5 33.4 18 48.45 14. 1 14. 3 34. 2 19 52. 72 15.33 15. S 35. 8 20 46. 61 13. 55 14. 3 34. 0 21 52. 52 15. 28 13. 9 34.4 22 50. 69 14. 74 15. 0 35. 2 23 48.45 14. 09 14. 0 35. 0 24 31.55 9.18 9. 8 27. 6 25 41. 32 12. 01 12. 1 32. 8 26 40. 92 ll. 91 12.9 32. 4 27 49. 87 14. 51 15. 0 35. 2 28 50. 28 14. 62 15. 2 36. 2 29 46. 81 13. 61 14. 3 35. 0 30 49. 87 14. 51 14. 7 34. 8

1 Iron levels are suspect. In each case this suspect result was obtained after running a series oi Fe standards and could possibly be due to incom plete flushing of sample inlet system.

N ores:

(A).Iron content was determined by atomic absorption spectros copy. The blood was diluted with 3 molar acetic acid and the absorption determined at 372A. against standard solution of On storing, a solution of perchloric acid (approximately 0.003% w./v.) and (3.1. Solvent Blue 19 (approximately 0.001% w./v.) in glacial acetic acid, precipitation was observed to occur. Investigation revealed that the Cl. Solvent Blue 19 contained two major components which can be separated by thin-layer chromatography and that one of these components is stable in a solution of perchloric acid in glacial acid whereas the other is relatively unstable yielding the black precipitate. The stable component has been named Abbott Blue and may be prepared from commercial C.I. Solvent Blue 19 by thicklayer chromatography on Silica Gel G using toluene as the eluent. Abbott Blue occurs as the band of highest Rf value of the two main components. Abbott Blue has been found to have the following characteristics:

(i) 0.1 mg. dissolved in a solution of perchloric acid (approximately 0.006% in glacial acetic acid shows an absorption peak at 505 m (ii) 0.1 mg. dissolved in a solution of potassium hydrogen phthalate (approximately 0.0012%) in glacial acetic acid shows absorption peaks at 595 m and 640 m EXAMPLE 2 Preparation of standardization of the test reagent The complete preparation and standardization of the reagent was carried out in containers fabricated of a material which was unalfected by perchloric/ acetic acids and which did not affect the strength of the test reagent.

(i) Preparation.A pilot batch of 7.5 litres of reagent was prepared in a 10 litre round bottom glass vessel. The vessel was first washed with approximately 500 m1. of glacial acetic acid which was removed and discarded. 7.5 litres of reagent grade glacial acetic acid were then put into the flask together with 75 mg. of Abbott Blue. The mixture was stirred for ten minutes to dissolve the dye. Fifty ml. of this solution were removed and titrated with N/lOO perchloric acid. The end-point was checked by measuring the absorption. The amount of N/ 10 perchloric acid required to neutralize the bulk liquid remaining in the vessel was calculated and added. A further 48.9 ml. of N/ 10 perchloric in acetic acid were added and stirred for a further ten minutes.

(ii) Assay of bulk test reagent-Fifty grams of the test reagent were pipetted into a glass titration vessel of approximately dimensions 5" x 1%" diameter containing a glass coated magnetic stirring bar and titrated with a standard solution of potassium hydrogen phthalate using a calibrated automatic recording titrimeter coupled with a photometric or spectrophotometric end-point detector.

(iii) Abbott Blue content in bulk 1iquid.'1"he light absorption curve of the bulk liquid had a maximum at 505 11111..

(iv) Fill weight of test reagent into kit.-

where H=selected haemoglobin concentration (g./ ml. whole blood) corresponding to the mid-point of the colour change.

V=volume of blood (microlitres) to be added to the test reagent kit.

C=concentration (mg/100 g.) of perchloric acid in the bulk test reagent.

(v) Container for test reagent kit.--'Il1e test reagent is filled into containers which are not affected by the reagent and themselves do not aifect the reagent. Neutral glass bottles prewashed with dilute acid and dried, closed with polytetrafiuoroethylene discs set in a screw cap, constitute a satisfactory system.

Stability of test reagent Test solutions of this type do not discolour when protected from direct light, i.e., in dark rooms or in suitable Haemoglobin leveloxyhae- Test moglobin, g./ kit Number 100 ml. result 11 211 211 2 271131 020 0121022 2 301309727111 mmllmlllmmmlllfl lfil 1111 U111B1111111 1HIM11111 1 111 TABLE 3 Haemoglobin Ieveloxyhae- Test moglobin, g./ kit 100ml. result Number cartons, and are stable for three months or more at room temperature.

EXAMPLE 3 The test kit NOTE.| denotes pink colour obtained; denotes blue colour ob tained; denotes intermediate colour obtained.

As seen above, the procedure required for the determination of a pre-selected amount of haemoglobin in a blood sample is extremely simple and fast and enables the consulting physician to quickly determine whether the patients blood is above or below the set level. In the above 50 examples, the test solution, its amount and the size of the blood sample are balanced in such a manner that the screening tests show the presence or absence of that haemoglobin level which represents 80% of the haemoglobin content normally encountered. It will be obvious to those 55 skilled in the art that proper adjustments of size of blood sample, volume of test solution, or concentration of test solution can be made to get a reading for the presence of 90% of the theoretical amount of haemoglobin; all

5 4 t mam Tnmr nym no 635282527547899753839008190237 i om wwmwfl. MMBMENMEMHEM MBMBMMMBMM BHMHMM l lg 2. 6 $3 a 1m 8 y f H c m TABLE 2 The test kit for carrying out the aforesaid screening test comprises packs containing a number of, say 10, bottles of reagent together with a number of, say 12, calibrated capillaries for blood measurement. The test is carried out as follows:

Fill the capillary tube with freshly drawn whole blood microlitres of blood) and drop into a bottle of the test reagent; tightly capped. Shake vigorously for 10-15 seconds and read at once. If the solution remains pink, the patient has less than 12 g./ 100 ml. (approximately 80%) haemoglobin. If it turns blue, the patient has more than 12.7 g./100 ml. (approximately 85%) haemoglobin. The reagent consists of an accurately standardized solution of perchloric acid in glacial acetic acid plus an indicator.

Test kits, containing in each bottle 0.640 mg. perchloric acid and 0.1 mg. (approximately) Abbott Blue in approximately 10 ml. glacial acetic acid, were used to test individual samples of freshly drawn blood of 20 microlitres each. The haemoglobin content of these 30 blood samples was also determined by the cyanmethaemoglobin method. The comparative results are depicted in Table 2.

Number the above parameters can be adjusted to suit other levels or allow the necessary adjustments for populations in which the normal haemoglobin content diifers materially from the levels discussed above; for instance, people living at high altitudes have a different a verage haemoglobin content in their blood, and the above test can obviously be adjusted easily to such a population.

EXAMPLE 4 The above test not only produces quick and accurate results for the screening of haemoglobin levels, but it also requires no particular skill by the practitioner: there are no samples to be diluted or adjusted and the test can be run with very minute amounts of blood, as for instance obtained by the finger-pinch method. In particular, the blood sample does not have to be treated with anti-coagulants or other adjuvants and does not require dilution; in fact, in order to obtain accurate and reproducible re- 75 sults, care should be taken that no measurable amounts I No'rE.-+ denotes'pink colour obtained; denotes blue colour obtained denotes intermediate colour obtained.

Test kits were prepared containing in each bottle 0.320 mg. perchloric acid and 0.1 mg. (approximately) C.I. Solvent Blue 19 in approximately 10 ml. glacial acetic acid and used to test 107 samples of freshly drawn blood (10 microlitres). The haemoglobin content was also determined by the hospital supplying the blood samples using the oxyhaemoglobin method. The results are depicted in Table 3.

of water enter the test vessels. The test vessels may be simple test tubes or bottles of clearly transparent glass or plastic material. In either instance, it is easy to provide vessel with graduation marks up to which the vessel is filled from the supply bottle containing the test solution. Graduation can be marked in such fashion that readings of +l0%, +20%, 10% and -20% of the norm can be used in connection with identical blood sample amounts.

What we claim is:

1. A method of determining haemoglobin in blood comprising admixing a sample of Whole blood with a solution of perchloric acid in glacial acetic acid, the amount of perchloric acid present corresponding to that amount necessary for the complete neutralization of a selected hae- References Cited UNITED STATES PATENTS 3/1968 Noller, H. G 23-230 B10 OTHER REFERENCES Lillie, R. D., Histopathologic Technic' and Practical Histochemistry, 3rd edition,pp. 147-8 (1965).

MORRIS'O. WOLK, Primary Examiner E. A. KATZ, Assistant Examiner US. Cl. X.R. 252408 

